以羊膜为底物培养鸡角膜缘上皮细胞的实验研究

目的为眼表面重建术提供良好的移植材料。方法用胰酶及EDTA消化法去除羊膜上皮将消化、离心获得的鸡角膜缘上皮细胞调成1×107/mlDMEM悬液,接种在6孔培养板底的羊膜基底膜面,放人37℃,5％CO2孵箱培养。结果48h羊膜上培养细胞形成单层,较培养板上培养的同种细胞小,细胞表面的微绒毛及侧面足状突丰富,立体感明显。结论以羊膜基底膜为底物培养的鸡角膜缘上皮细胞可以着床生长并形成完整的单层上皮,为眼表重建使用羊膜及基底膜面培养的角膜上皮细胞提供了实验材料。同时表明羊膜在体外也可以促进鸡角膜缘上皮细胞生长。     

羊膜培养兔角膜缘上皮细胞及自体移植--角膜缘干细胞缺损的治疗

目的　探讨以人羊膜为载体培养兔角膜缘上皮细胞及其自体移植治疗全角膜缘干细胞缺损。方法　在8只兔右眼用正庚醇脱上皮和角膜缘环切的方法构建全角膜缘干细胞缺损模型2月。其中6只兔为实验组,活体取左眼角膜缘浅层小块,置羊膜上常规和气_液培养42天后进行自体移植治疗右眼角膜缘干细胞缺损;2只兔为对照组,直接用解冻无细胞人羊膜移植治疗右眼角膜缘干细胞缺损。进行细胞和术眼活体观察、组织学观察和电镜观察。结果　角膜缘上皮细胞在羊膜上生长良好,形成复层,细胞间的联结结构存在,细胞与羊膜组织粘附牢固。实验组移植手术后角膜迅速上皮化,恢复角膜表面光滑和透明,组织学观察和电镜观察呈现生理角膜上皮层的结构特点。但眼睑闭合不全可导致手术失败。对照组术后出现角膜缘干细胞缺损导致的角膜病变。结论　以羊膜为载体培养角膜缘上皮细胞后自体移植可有效地治疗角膜缘干细胞缺损导致的角膜病变。     

羊膜为底物角膜缘上皮细胞培养方法的探讨

目的　探讨在羊膜上培养角膜缘上皮细胞 (limbal epithelial cells,LEC)的合适方法。方法　切取大小约 2 mm× 2 mm、厚约 2 0 0 μm含有完整上皮细胞的兔角膜缘组织 ,剪切成 4个 1 mm× 1 mm小的组织块 ,以羊膜为底物分别使用组织块培养法、组织块培养后传代培养法和组织块经消化酶处理后培养法培养兔 L EC,通过倒置显微镜、细胞组织学和扫描电镜观察细胞生长情况。结果　使用上述 3种方法培养的 L EC均可在羊膜上形成密集单层。细胞组织学检查显示在羊膜上培养的 LEC尚可形成多层。扫描电镜观察 LEC立体感强、细胞表面微绒毛丰富。但以组织块经消化酶处理后培养法培养 L EC较为快速。结论在羊膜上上述 3种方法都可用于 LEC的培养 ,但以组织块经消化酶处理后培养法更为实用     

羊膜为载体的人角膜缘上皮细胞移植治疗大鼠角膜碱烧伤的研究

目的研究人角膜缘上皮细胞的生物学特性,探讨体外培养的人角膜缘上皮细胞移植进行眼表重建的临床应用价值及前景。方法体外培养人角膜缘上皮细胞,通过免疫荧光染色法和逆转录聚合酶链反应（RT．PCR）法分析表型,Brdu掺入法检测慢周期（slow—cycling）细胞。建立大鼠角膜碱烧伤模型,随机分为单纯对照组、羊膜移植（AMT）组及羊膜为载体的角膜缘上皮细胞移植（LSAT）组,术后通过裂隙灯显微镜观察、角膜切片HE染色及免疫荧光染色等方法对各组大鼠眼表恢复情况进行评价。结果培养早期大多数角膜缘上皮细胞p63和K19染色阳性,仅少数细胞K3染色阳性;随培养时间的延长,K3阳性细胞增多,部分细胞同时表达p63和K3。RT—PCR示培养的角膜缘上皮细胞中p63和K12均有阳性表达,而角膜上皮细胞仅表达K12。Brdu掺入法检测到慢周期细胞。动物实验表明,LSAT可以显著减轻角膜病变,使角膜恢复完整性和透明性,眼表评分各项指标与AMT组和对照组比较差异均有统计学意义。LSAT组大鼠角膜切片染色示大部分上皮细胞抗人核和K3染色阳性。结论体外培养的人角膜缘上皮细胞中p63不仅表达于角膜缘干细胞,在短暂扩充细胞中也有一定量的表达;慢周期细胞的存在证实了培养的角膜缘上皮细胞中角膜缘干细胞的存在,p63和K19阳性、K3阴性的细胞为角膜缘干细胞;羊膜为载体的体外培养的人角膜缘上皮细胞移植可有效重建眼表,具有较高的临床应用价值与发展前景。     

兔角膜缘上皮细胞培养后自体移植修复

目的：运用培养角膜缘上皮细胞联合人羊膜行自体移植的方法，观察植片修复兔眼角膜上皮的疗效。方法：选用健康新西兰白兔20只，制成右眼角膜缘干细胞缺乏的兔眼模型，其中12只兔行角膜缘上皮细胞培养联合羊膜自体移植，另外8只兔只进行单纯羊膜移植。术后每周对眼表情况进行评分，术后1mo眼角膜进行苏木素-伊红(HE)染色和透射电镜观察。结果：移植了含有自体角膜上皮细胞的兔眼，术后早期都形成了角膜上皮化并明显抑制了新生血管的再生，HE染色和电镜观察表明培养并移植的角膜上皮与正常的角膜上皮无明显差异；而只接受羊膜移植的兔眼，术后又出现角膜混浊和明显的新生血管，表明角膜表面被结膜上皮覆盖。结论：该方法术后早期可以恢复角膜上皮化，重建正常眼表，疗效明显优于单纯羊膜移植。     

不同体外方法培养兔角膜缘上皮细胞作用的研究

自从1986年Schermer等发现角膜缘干细胞位于角膜缘基底Vogt栅栏内后，对角膜缘干细胞缺失和角膜上皮病变治疗有了许多新的疗法，其中以角膜缘上皮细胞体外培养法治疗上述疾病比较有效。目前，多数学者采用组织块法进行角膜缘上皮细胞体外培养，但在培养系中干细胞能否从组织块中迁移出来尚有争议。另外，最近角膜缘上皮单细胞悬浮液超低温冷藏、复苏以及体外培养技术越来越受到人们的关注，成为角膜缘干细胞研究中的一项重要的课题。此种培养方法既可以将角膜缘干细胞和其他角膜上皮细胞长期保存在超低温状态下，减少细胞传代培养次数，[第一段]     

聚乙二醇水凝胶膜为载体的人角膜上皮细胞原代培养研究

目的 观察人角膜上皮细胞（corneal limbal epithelial cells,CLEC）的生物学特性,探讨CLEC在普通培养皿和聚乙二醇水凝胶膜（poly(ethylene glycol)-based hydrogel films,PHF）上的增殖率差异。设计 实验研究。研究对象 人角膜上皮细胞。方法 采用组织块培养法体外扩增CLEC,待细胞生长至指数生长期,分别种植于普通培养皿和PHF上,倒置显微镜下观察培养细胞的生长特点,利用免疫荧光对其鉴定,利用CCK-8比色法观察两组细胞相对增殖率（relative growth rate, RGR）的差异。主要指标 光镜下CLEC的生长,AE1染色,CCK-8比色法的吸光度（optical density,OD）值。结果 光镜下可见CLEC在普通培养皿和PHF上均能移行扩增、融合成片,在PHF上的生长速度与普通培养皿组相近。两组细胞AE1荧光染色均呈阳性,PHF组阳性率（73.26%±8.84%）与普通培养皿组（70.84%±3.51%）基本相同。PHF组在12 h（0.89±0.06）、24 h（1.13±0.10）、48 h（1.24±0.03）的OD值与普通培养皿组（0.89±0.03、1.08±0.04、1.28±0.09）差异无统计学意义（P=0.79,0.36,0.76）。PHF组细胞在12 h（99.12%±4.81%）、24 h（103.74%±5.55%）、48 h （97.83%±13.37%）的RGR均大于75%,各时间点间差异无统计学意义（P=0.37,0.90）。结论  PHF可以作为体外扩增角膜上皮细胞的良好载体。（眼科, 2016, 25： 405-408）     

角膜缘移植术

角膜表面的正常与稳定，对于保持角膜的透明、维持其正常的生理功能是相当重要的。角膜表面由于其解剖位置的关系而容易受到各种各样的损害，较严重的损害，往往导致持续性角膜上皮糜烂，新生血管侵入及假性胬肉形成。而这些又是角膜病致盲及角膜移植失败的主要原因[1，2]。为了修复受损角膜表面．人们进行了一系列的研究。角膜线移植术（limbaltransplamtation，LT）是近年发展的、用于修复角膜表面的一种手术方法。其临床应用取得了较好的效果。一、角膜缘移植术的有效机制于细胞存在于所有自我更新的组织，干细胞是自我更新组织内细胞增…     

角膜缘移植重建角膜表面

角膜表面的正常与稳定，对于维持角膜的透明相当重要。各种原因所造成的角膜上皮糜烂，新生血管侵入及假性胬肉形成是角膜病致盲和角膜移植失败的主要原因。     

培养的兔角膜缘上皮细胞深低温保存后的生物学活性研究

目的探讨深低温保存角膜缘上皮细胞的可行性和效果。方法将兔角膜缘组织培养出的细胞,分别用两组冷冻保护液冻存,0.5、1、2个月后分批取出。将未冷冻的细胞和不同冻存时间、不同冻存保护液的冷冻复苏后细胞传代培养,采用免疫组化、电镜鉴定培养后细胞的性质。通过倒置显微镜、电镜、MTT比色法来比较传代培养的角膜缘上皮细胞深低温保存前后的形态结构和生物学活性。结果AE1和传代细胞鼠抗兔增殖细胞核抗原(PCNA)单克隆抗体染色呈阳性反应,AE5单克隆抗体染色偶见阳性反应;冻存后的传代细胞贴壁、生长均滞后,而且电镜检查均有不同程度细胞水肿,再培养7d后形态结构均恢复正常;MTT法检测复苏当天冻存后的细胞比未冻存的细胞增殖活性低,差异显著(P<0.05,但培养5d后无显著差异(P>0.05),甘油组和DMSO组间有显著性差异(P<0.05),不同冻存时间的各冻存组测得值无显著差异(P>0.05)。结论冻存的角膜缘上皮细胞传代培养仍保持上皮细胞特性并含有干细胞;DMSO保护液比甘油液低温保存效果好;角膜缘上皮细胞经阶段降温后深低温保存简单可行,选择适当的冷冻保护剂冻存后的细胞再培养,其细胞活性和结构与原代相似。     

Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane

PURPOSE: Surgery to reconstruct the ocular surface is greatly facilitated by the use of amniotic membrane, either as a biologic drape or, more recently, as a substrate for the transplantation of cultivated corneal epithelial cells. This study was designed to compare the usefulness of intact and denuded human amniotic membranes as a substrate for corneal epithelial cell culture. METHODS: Small (3-mm-diameter) biopsy specimens of superficial cornea including epithelium were excised from the central and limbal regions in rabbits. They were cultured on human amniotic membrane with or without amniotic epithelial cells and examined by light, scanning electron, and transmission electron microscopy. RESULTS: Cellular outgrowth from the central explants (n = 10) after 14 days in culture measured 1.82 +/- 2.62 mm2 on intact amniotic membrane and 131.83 +/- 28.31 mm2 on denuded amniotic membrane. In contrast, outgrowths from the limbal explants (n = 10) at the same time measured 4.58 +/- 4.56 and 505.39 +/- 134.20 mm2 on intact and denuded amniotic membranes, respectively. The leading edges of the outgrowths on intact amniotic membrane were much less uniform than those on denuded amniotic membrane, and, in the former, corneal epithelial cells appeared to migrate over the top of amniotic epithelial cells. Limbal cells cultivated on denuded amniotic membrane formed a nicely stratified layer that adhered well to the underlying amniotic membrane. CONCLUSIONS: Denuded amniotic membrane appears to be an excellent substrate for the cultivation of corneal epithelial cells, with a view to transplantation.     

Evaluation of corneal damage caused by iodine preparations using human corneal epithelial cells

Purpose

To compare the cytotoxicity of povidone–iodine solution (PVP-I) with that of polyvinyl alcohol–iodine solution (PAI) for ophthalmic use.

Methods

Cells of the human corneal epithelial cell line HCE-T were exposed to various dilutions of PVP-I or PAI, and the cytotoxicity of these two antiseptics was evaluated. The relative toxicities of PVP-I and PAI were also investigated following the inactivation of iodine by achromatization with sodium thiosulfate.

Results

PVP-I and PAI had equivalent antiseptic effects, but the cytotoxicity of PVP-I diluted 16-fold was higher than that of PAI diluted 6-fold. Following inactivation of iodine with sodium thiosulfate, the cytotoxicity of PVP-I remained concentration dependent, whereas PAI exhibited a low toxicity that was similar to the effect of saline on cell viability. Exposure to lauromacrogol, a surfactant used in PVP-I, in solution at concentrations of 1–1000 mg/mL clearly resulted in corneal cytotoxicity. The PVP-I and PAI solutions had a pH value of 4.0 and 5.2, respectively. HCE-T cells were significantly more viable at pH 7 than at pH 1–6.

Conclusion

To avoid corneal damage, preoperative antisepsis of the surgical field should be accomplished with PAI diluted 6-fold, rather than with PVP-I diluted 16-fold. The toxicity of the iodine compound stems primarily from the available iodine concentration and partly from its pH, surfactant and osmolality. Further clinical investigations are required in order to determine the optimal concentrations for use.     

Keratin filaments of corneal epithelial cells

Intermediate filaments with a diameter of approximately 8–10 nm are present in great abundance in the cytoplasm of corneal epithelial cells. In order to study the immunological relationship between these filaments and the tonofilaments of epidermal cells, we prepared an antiserum against keratin proteins isolated from the stratum corneum of human epidermis. Immunofluorescent staining of frozen sections of rabbit and human corneas with this anti-keratin antiserum resulted in the intense staining of the entire corneal epithelium, but no staining of the endothelium or any of the stromal components. Similar staining of cultured rabbit and human corneal epithelial cells revealed a cytoplasmic network of wavy fibers distinct from microfilaments and microtubles. Electron microscopic examination of cultured rabbit corneal epithelial cells showed that they formed a stratified squamous structure with prominent desmosomal cell-cell junctions and wavy bundles of cytoplasmic 8–10 nm filaments. These results suggest that the 8–10 nm filaments of corneal epithelium are immunologically related to tonofilaments of the epidermis and are composed of keratin proteins. Since corneal epithelium is the only keratin-containing cell type in normal cornea, anti-keratin staining provides a simple, specific and sensitive method for identifying corneal epithelial cells. Further characterization of the keratins synthesized by corneal epithelial cells under various in vivo and in vitro conditions may provide useful information concerning the mechanisms of corneal epithelial differentiation.     

Injured corneal epithelial cells promote myodifferentiation of corneal fibroblasts

PURPOSE: To determine whether injured corneal epithelial cells stimulate myodifferentiation in corneal fibroblasts and whether transforming growth factor (TGF)-beta is involved. METHODS: Rabbit corneal fibroblasts were cultured on collagen gel, with or without cocultured corneal epithelial cells or with partially scraped epithelial cells, on a companion plate separated by a permeable membrane. To evaluate fibroblast-induced gel contraction, gel thickness was measured daily relative to the original thickness. Total fibroblasts on the gel were counted. Myofibroblasts were counted by using immunocytochemical identification with anti-alpha-smooth muscle actin (alpha-SMA). TGF-beta was assayed in the media on days 3 and 6. These procedures also were performed in the presence of anti-TGF-beta antibody. RESULTS: Gel contraction, alpha-SMA-positive cells, and total cell number were significantly greater on gels with injured epithelial cells than on gels without epithelial cells or with uninjured epithelial cells, as was TGF-beta concentration in the media. Anti-TGF-beta antibody eliminated these differences. CONCLUSIONS: Injured epithelial cells stimulate myodifferentiation in fibroblasts through one or more soluble factors, including TGF-beta.     

Longterm survival of transplanted human corneal epithelial cells and corneal stem cells

PURPOSE: To investigate the survival of donor-derived epithelial cells in conventional penetrating keratoplasty (PKP) and in homologous penetrating central limbal keratoplasty (HPCLK). METHODS AND PATIENTS: Epithelial cells from 26 eyes of 26 patients were analysed. All cases were sex-mismatched (i.e. the transplant and patient were of different genders). At suture removal more than 1 year post surgery, epithelial cells were obtained by gently wiping the removed sutures on glass slides. The cell samples were analysed using fluorescent in situ hybridization (FISH) of the sex chromosomes. This technique makes it possible to allocate the origin of each cell nucleus to either the donor or the recipient. RESULTS: All 19 conventional PKPs were clear and seven had donor-derived epithelial cells at suture removal. Five of the seven HPCLK grafts were clear at the time of investigation (365-1355 days post surgery), and donor-derived epithelial cells were found in two grafts. CONCLUSION: Harvesting cells from removed sutures in combination with FISH enables the clinical study of cell survival in corneal transplants without jeopardizing functioning grafts. From the limited sample investigated, the following tentative conclusions can be made. Donor-derived epithelial cells can remain in conventional PKP for over 1 year. In combined stem cell and corneal grafts (HPCLK), donor-derived epithelial cells may also be retrieved at 1 year or beyond following surgery but the correlation between their presence and a remaining clear graft is uncertain.     

Longterm survival of transplanted human corneal epithelial cells and corneal stem cells

PURPOSE: To investigate the survival of donor-derived epithelial cells in conventional penetrating keratoplasty (PKP) and in homologous penetrating central limbal keratoplasty (HPCLK). METHODS AND PATIENTS: Epithelial cells from 26 eyes of 26 patients were analysed. All cases were sex-mismatched (i.e. the transplant and patient were of different genders). At suture removal more than 1 year post surgery, epithelial cells were obtained by gently wiping the removed sutures on glass slides. The cell samples were analysed using fluorescent in situ hybridization (FISH) of the sex chromosomes. This technique makes it possible to allocate the origin of each cell nucleus to either the donor or the recipient. RESULTS: All 19 conventional PKPs were clear and seven had donor-derived epithelial cells at suture removal. Five of the seven HPCLK grafts were clear at the time of investigation (365--1355 days post surgery), and donor-derived epithelial cells were found in two grafts. CONCLUSION: Harvesting cells from removed sutures in combination with FISH enables the clinical study of cell survival in corneal transplants without jeopardizing functioning grafts. From the limited sample investigated, the following tentative conclusions can be made. Donor-derived epithelial cells can remain in conventional PKP for over 1 year. In combined stem cell and corneal grafts (HPCLK), donor-derived epithelial cells may also be retrieved at 1 year or beyond following surgery but the correlation between their presence and a remaining clear graft is uncertain.     

An evaluation of cultivated corneal limbal epithelial cells,using cell-suspension culture

PURPOSE: A previous report has described an ocular surface reconstruction method involving the use of cultivated corneal epithelium derived from limbal explants. In the current study, a new culture system was developed involving the in vitro propagation on amniotic membrane (AM) of epithelial cells from enzymatically dissociated limbal epithelium. The purpose of this new method is to produce a cultivated epithelial cell layer that contains stem cells and that is superior to explanted cultivated epithelium. The new cell-suspension technique was compared with the existing explant method. METHODS: Limbal epithelial cells were dissociated from donor eyes by dispase and seeded on the denuded AM. Small pieces of limbal epithelium were also cultured on denuded AM as explant cultures. The cultivated epithelium was examined by electron microscopy and immunohistochemistry for cornea-specific keratins (K3 and K12). RESULTS: Both cell-suspension and explant culture methods produced a healthy epithelial cell layer. The cell-suspension culture had significantly (P < 0.001) more desmosomal junctions between the explant-cultured basal cells. In addition, the intercellular spaces between the cell-suspension's basal cells were significantly (P < 0.001) smaller than those between the explant-cultured basal cells. Both types of cultivated epithelium showed positive expression of K3 and K12 keratins. In the cell-suspension culture, expression of K3 and K12 keratins was more prominent in the superficial cells. CONCLUSIONS: Corneal epithelial cells were successfully regenerated in vitro by a cell-suspension culture system. The suspension-cultured epithelium must include some stem cells and morphologically is significantly superior to explant-cultured epithelium. Thus, this new technique is potentially more suitable for cultivated corneal limbal epithelial transplantation.     

Bacterial invasion of corneal epithelial cells.

PURPOSE: The normal ocular surface is frequently colonized by commensal gram-positive species. Gram-negative bacteria are often implicated in corneal infection and inflammation, particularly in association with soft contact lens wear. The aim of this study was to elucidate possible mechanisms of virulence in ocular bacteria. METHODS: The susceptibility of a human corneal epithelial cell line to bacterial invasion and association was evaluated using the gentamicin exclusion assay. Organisms tested included isolates from corneal ulcers, corneal inflammation and ocular sites in asymptomatic individuals. RESULTS: The commensal, non-pathogenic bacterium Staphylococcus epidermidis and some pathogenic strains of Serratia marcescens did not invade corneal epithelial cells. In contrast, pathogenic strains of Pseudomonas aeruginosa associated with and invaded corneal epithelial cells. CONCLUSIONS: The increased association of P. aeruginosa, compared to other bacterial types, might be a reason for the more frequent association of this bacterium with contact-lens-associated microbial keratitis.     

人角膜上皮干细胞的识别

目的 探讨人角膜上皮干细胞的分子标记。方法 对人角膜和角膜缘部位行组织学检查以分析角膜缘解剖结构。对人角膜切片和整个角膜组织行免疫组织化学染色以检测中央角膜和角膜缘部位未分化标记,如核蛋白p63、乳腺癌抵抗蛋白（ABCG2,BCRP1）、烯醇化酶α、整合素拍、胡及β1、表皮生长因子受体（EGFR）、细胞角蛋白19（CK19）、14（CK14）及转铁蛋白受体（CDT1）的表达,经荧光显微镜和激光扫描共焦显微镜观察。对角膜中部和角膜缘上皮细胞的mRNA进行半定量逆转录聚合酶链反应（RT—PCR）和原位杂交以检测其相关基因的表达。结果 角膜缘部位横向切片显示角膜缘上皮细胞为乳头放射状排列,对应于Vogt栅栏环境。未分化标记整合素β1、EGFR、烯醇化酶α及CK19在角膜缘基底细胞胞质染色较表层细胞更强;p63、ABCG2、整合素胡蛋白仅见于角膜缘基底部上皮细胞。激光扫描共焦显微镜观察和RT—PCR结果显示角膜缘表达p63、ABCG2、整合素胡蛋白及mRNA。原位杂交显示p63仅表达于角膜缘基底层细胞。结论 角膜缘上皮呈乳头放射状排列,角膜缘干细胞群具有复合标记：p63表达于细胞核、ABCG2表达于胞质、整合素胡表达于胞膜。采用这些标记复合体,可将角膜缘干细胞群与其他上皮细胞区分。     

Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells

PURPOSE: In this study we established a protocol for transfection of human corneal endothelial and human retinal pigment epithelial cells. This protocol was used for immortalization of human corneal endothelial cells. METHODS: Transfection was performed by means of electroporation. For immortalization a plasmid encoding large and small SV40 T-antigen was used. RESULTS: The established electroporation protocol was suitable for both cell types. This protocol was used for transfection of human corneal endothelial cells with a plasmid containing the early region of SV40. The transfected cultures exhibited an increased life-span before they entered crisis. One culture recovered from crisis and was cultivated for 300 population doublings. The cells exhibited an in vivo-like morphology usually lost during cell culture. CONCLUSIONS: We describe for the first time a culture of SV40 transfected human corneal endothelial cells which recovered from crisis and can therefore be regarded as immortalized.